1. Technical field
The present invention relates to a complex comprising hepatocyte growth factor (HGF) and the met proto-oncogene protein. The present invention also relates to a method for detecting the presence of HGF ligand or met proto-oncogene receptor and to a method for isolating either the ligand, the receptor or a complex comprising both.
The present invention further relates to methods of diagnosing and treating conditions proliferative disorders such as hepatitis, hepatocarcinogenesis, carcinogenesis and wound healing.
Further, the present invention relates to a method of stimulating the proliferation of hemtopoietic progenitors with HGF.
2. Background of the Invention
Hepatocyte growth factor (HGF) was first purified from human and rabbit plasma and rat platelets on the basis of its ability to stimulate mitogenesis of rat hepatocytes (Gohoda et al. J. Clin. Invest. 81, 414 (1988); Zarnegar et al. Cancer Res. 49, 3314 (1989); Nakamura et al. FEBS Lett. 224,311 (1987)). Thus, HGF may act as a humoral factor promoting liver regeneration after partial hepatectomy or liver injury (Michalopoulos FASEB J. 4, 176 (1990)). The same factor was purified from human fibroblast culture medium and shown to act on melanocytes and a variety of epithelial and endothelial cells (Rubin et al. Proc. Natl. Acad. Sci. USA 88, 415 (1990)). Together with evidence of HGF expression in several organs (Rubin et al. Proc. Natl. Acad. Sci. USA 88, 415 (1990); Tashiro et al. Proc. Natl. Acad. Sci. USA 87, 3200 (1990); Zarnegar et al. Proc. Natl. Acad. Sci. USA 87, 1252 (1990); Kinoshita et al. Biochem. Biophys. Res. Comm. 165, 1229 (1989)), these findings indicate that HGF may also act as a paracrine mediator of proliferation for a broad spectrum of cell types. Molecular cloning of HGF revealed a remarkable structural homology to plasminogen and related serine proteases (Rubin et al. Proc. Natl. Acad. Sci. USA 88, 415 (1990); Nakamura et al. Nature 342, 440 (1989); Miyazawa et al. Biophys. Res. Comm. 163, 967 (1989)). Recent evidence that HGF induces rapid tyrosine phosphorylation of proteins in intact target cells suggests that a tyrosine kinase receptor might mediate its mitogenic signal (Rubin et al. Proc. Natl. Acad. Sci. USA 88, 415 (1990)).
HGF is structurally related to the family of serine proteases that includes plasminogen, prothrombin, urokinase, and tissue plasminogen activator (Rubin et al. Proc. Natl. Acad. Sci. USA 88, 415 (1990)); Nakamura et al. Nature 342, 440 (1989)). As defined herein, HGF includes a variant of HGF previously characterized as a broad-spectrum mitogen called plasminogen like growth factor (PLGF). Several proteases, including members of the serine protease family, stimulate DNA synthesis presumably through a proteolytic mechanism similar to tryptic activation of the insulin receptor (Shoelson et al. J. Biol. Chem. 263, 4852 (1988)). To date, only urokinase has been found to associate with a specific cell-surface receptor, which itself bears no homology to any known tyrosine kinase receptors (Roldan et al. EMBO J. 9, 467 (1990)). It has recently been observed that HGF is very similar or identical to scatter factor (Gherardi, et al. Nature 346, 28 (1990); Gherardi, et al. Cancer Cells 3, 227-232 (1991); Weidner, et al. Proc. Nat. Acad. Sci. USA 88, 7001-7005 (1991)). Scatter factor has been characterized as causing epithelial cells to dissociate from each other and begin migration (Gherardi, et al. Proc. Nat. Acad. Sci. USA 86, 5844-5848 (1989); Weidner, et al. J. Cell Biol. 111, 2097-2108 (1990); Stoker, et al. Nature 327, 239-242 (1987 )). These observations suggest that HGF might function in the growth and renewal of epithelial cells that would be required in wound repair. Further, under proper conditions, HGF may have a stimulatory, as well as inhibitory, effect on hematopoietic cells.
It is clear that a need exists to identify the receptor of HGF. The present invention provides such a receptor, which receptor is the met proto-oncogene product, and a complex comprising HGF and met proto-oncogene protein. The met proto-oncogene protein is a member of the tyrosine kinase growth factor receptor family.
cMet mRNA has been detected in several murine myeloid progenitor tumor cell lines (Iyer et al. Cell Growth and Diff. 1, 87-95 (1990)), raising the question whether HGF might be mitogenic for these cell lines. The effect of HGF on the growth and inhibition of one such cell line, NFS-60, has been examined. The NFS-60 cell line requires IL-3 to maintain its growth in vitro and is representative of an immature hematopoietic progenitor blocked in differentiation (Holmes et al. Proc. Nat. Acad. Sci. USA 82, 6687-6691 (1985); Hara et al. Exp. Hematol. 16, 256-261 (1988)). It has previously been shown to express significant levels of met mRNA (Iyer et al. Cell Growth and Diff. 1, 87-95 (1990)). The ability of HGF to stimulate [.sup.3 H]thymidine incorporation into NFS-60 cells was examined.
These findings, combined with the previously documented effects of HGF upon hepatocytes, epithelial cells, endothelial cells, and melanocytes, taken with the apparent close relationship between scatter factor and human growth factor, demonstrate that HGF is a growth factor for renewable cells from a variety of tissues. The ability of HGF to stimulate both liver regeneration and myeloid progenitors is strikingly similar to the effects of IL-6, which has been shown to induce proliferation of hepatocytes (Kuma, et al. Immunobiol. 180, 235-242 (1990)), and to act as a synergistic factor for IL-3 dependent colony formation (Ikebuchi, et al. Proc. Natl. Acad. Sci. USA 84, 9035-9039 (1987); Leary, et al. Blood 71, 1759-1763 (1988)).
Knowledge of the receptor/ligand relationship involving HGF will facilitate the study and treatment of proliferative disorders in which expression of these molecules plays an important role. Additionally, identification of the met proto-oncogene receptor-HGF complex provides a means for identifying tissues other than liver tissue affected by factor binding.